In the central nervous system (CMS) of vertebrates, the vascular epithelium and closely apposed astrocytes are the physiologic barrier, known as the blood-brain barrier (BBB) that ensures a distinct neuronal cellular environment. At least two properties of the barrier influence the ability of substances to pass from the humoral compartment into the CNS: a diffusional barrier attributed to specialized lateralized cellular junctions in the epithelium, and the active transport of small molecules across the lipid bilayers of the cell layers. These two properties maintain the chemical separation of the compartments that protects the CMS from xenobiotics, but they also interfere with the entry of useful Pharmaceuticals into the brain. Currently, the methods for controlling therapeutic drug access to the brain are crude, inefficient, and/or dangerous in the clinical setting. Thus, safe and effective methods for controlling drug access to the CNS would have great impact on disease treatment and other clinical intervention in the CNS. We recently reported the discovery of a novel BBB regulatory system in Drosophila. This work suggests that the BBB is a dynamic structure that participates in regulating its own properties of exclusion. Our goal is to understand the regulatory mechanisms that integrate barrier properties and control access of small molecules to the CNS. Drosophila BBB anatomy has unique advantages for the simultaneous study of different BBB physiologies, specifically targeting the inter-relationship of humoral barrier epithelium and associated glia. We have shown that the properties of the Drosophila BBB are remarkably similar to those of the vertebrate BBB, and we have developed in vivo and quantitative methods for measuring the control of paracellular border function and drug transport. With these methods, we can integrate the study of physiology, anatomy, and genetics at the humoral-CNS interface and have a novel opportunity to understand the interdependence of the multiple layers of the BBB by looking simultaneously at function, pathology, and regulation. This is a powerful approach to better understand how the BBB might be manipulated for clinical purposes.